2.1 Tissue samples
A total of 40 wax blocks of patients with oral submucosal fibrosis (OSF) confirmed by pathology were selected from the Pathology Department of Haikou People's Hospital from January 2017 to December 2020, including 15 cases in the early stage, 13 cases in the middle stage and 12 cases in the late stage. None of the them underwent any preoperative treatment and were not associated with other systemic diseases or mucosal diseases. This research program has been permitted by the ethics Committee of Haikou People's Hospital.
2.2 Immunohistochemical (IHC) staining
The two-step IHC method was applied to assess the expression of NF-κB p65 and PDE4 in OSF (early, middle and late stage) tissue sections. After the dewaxing and rehydrating of paraffin sections, they were cleaned twice with PBS. Then the tissue antigen repair was performed, and the sections were blocked with a few drops of 3% H2O2. Furthermore, the sections were reacted with primary antibodies and secondary antibody successively, and DAB solution (Aike Reagent, China) was dropped for color-developing. All the antibodies used were purchased from Abcam, including: anti-NF-κB p65 (ab16502), anti-PDE4 (ab14613) and goat anti-rabbit IgG (ab97051). The slices were then washed with PBS, restained with hematoxylin (J&K Scientific, China), dehydrated and sealed for microscopic examination.
2.3 Cell culture
The OSCC cell lines CAL27 and HN30 were acquired from GuangZhou Jennio Biotech (China), and oral mucosal epithelial cells (OMES) were provided by Shanghai Baiyi Biotechnology Center (China). These two kinds of OSCC cells were all cultured in DMEM (Gibco, US) that containing 10% fetal bovine serum (Hyclone, US) and 1% double antibodies (Absin, China). The cell culture environment is constant 37℃ with 5% CO2.
Total RNA of CAL27 and HN30 cells was collected using Trizol (Keybio, China). Then the cDNA was obtained using the reverse transcription kit (Biopike, China). The following were sequences of hBD-3 and NF-κB p65 primers: hBD-3 F: 5’-TAGCCTAACGTAATCGACTG-3’, R: 5’-GACTAATGACCTACGTTCGAC-3’; NF-κB p65 F: 5’-GAGCTACATTGCAACTAGAC-3’, R: 5’-CTATGACCTACGACTGATCC-3’. The qPCR reaction system was constructed using 2× SYBR Green qPCR kit (Dingguo Changsheng, China), and the reaction was conducted and analyzed by Gentier 96E PCR analysis system (Tianlong, China). The tests were repeated independently for three times.
2.5 Western blot
CAL27 and HN30 cells were digested and lysed, and the protein of cell lysate was quantified using the BCA kit (Absin, China). Then 15 μg of total protein was injected into the sample holes for SDS-PAGE. Furthermore, the dividual proteins in the gel were transferred to the PVDF membrane (Absin, China). After blocking with 1% bovine serum albumin, the membrane was incubated overnight with diverse primary antibodies at 4℃. The antibodies were all provided by Abcam (UK): anti-hBD-3 (ab172703), anti-NF-κB p65 (ab288751), anti-IκB (ab97783), anti-c-Myc (ab152146), anti-p21 (ab227443), anti-GAPDH (ab9484). The second day, after incubating with secondary antibodies for 1 h, the protein bands could be visualized by developing.
2.6 Cell transfection
Oligonucleotide si-hBD-3 and si-NC was synthesized by Guangzhou Ribobio (China), si-hBD-3 F: 5’-UUCAUCGACCAUUACAGCUTT-3’, R: 5’-CAGUCACGUUCAGUGACAATT-3’, si-NC F: 5’-UGCAAGUUCAUGACUGATT-3’, R: 5’-GAUUACCGAUGACUAACGTT-3’. si-hBD-3 or si-NC was transfected into CAL27 and HN30 cells following the procedure of Lipofectamine™2000 Transfection Reagent (Invitrogen, US). For the overexpression of NF-κB p65, full length of NF-κB p65 was obtained by PCR amplification and inserted into the pcDNA3.1 plasmid (Miaoling, China) to construct the NF-κB p65 overexpression plasmid. Then the NF-κB p65- pcDNA3.1 or the empty pcDNA3.1 plasmid were transfected to CAL27 and HN30 cells using Lipofectamine™2000 reagent. Follow-up experiments could be performed 48 h after the transfection.
2.7 CCK-8 assay
This assay was carried out to assess the cell viability according to the working manual of Cell Counting Kit-8 (Sangon Biotech, China). Briefly, 5×105 of CAL27 or HN30 cells were inoculated in 96-well plates. When all cells were completely adherent to the plate, 10 μL of CCK-8 reagent was injected to treat cells for another 2 h. Finally, the OD450 was measured by the microplate reader (Tecan, Swiss).
2.8 Transwell invasion assay
The basement membrane of Transwell chamber (Corning, US) was pre-coated with Matrigel (Corning, US) one day before the experiment, and the chambers were seated in 24-well plates. Then the transfected CAL27 and HN30 cells were placed in the upper chamber, and 500 μL/well of the complete DMEM was injected into the lower chamber. After 24 h of culture, the chamber was taken out and stained with 0.1% crystal violet (Aladdin, China). Then the remaining cells on the upside of the basement membrane were erased with Q-tips, and the cells invaded to the bottom side were observed with the microscope (Nikon, Japan).
2.9 Statistical analysis
All quantitative data were shown as Mean ± Standard deviation. The SPSS software (V 19.0) was used for the statistical analysis. The comparison between two groups was conducted using Student’s t test, and one-way ANOVA was used for three groups. P < 0.05 was considered as statistically significant.